The present invention relates to novel molecules which may be advantageously incorporated in membrane based biosensors.
Previous patents such as WO 92/17788, U.S. Pat. No. 5,204,239 and WO 93/21528 (the disclosures of which are incorporated herein by reference) have described how functional biosensor bilayer or monolayer lipid membranes may be formed on a metal substrate such that a functioning ionic reservoir is formed between the metal surface and the lipid membrane. The inner leaflet of the membrane, or in the case of the monolayer membrane the whole membrane is typically assembled using molecules that comprise within the same molecule a hydrophobic group linked to a hydrophilic group onto which is attached an attachment group such as a disulfide or thiol group capable of attaching the molecule to an electrode. Furthermore, it has been disclosed in WO 94/07593 (the disclosure of which is also included herein by reference) that in order to provide improved reservoir characteristics and fluidity characteristics of the membrane a small spacer molecule, such as the disulfide of mercaptoacetic acid, should be incorporated between the reservoir molecules that had been adsorbed onto the metal surface.
The present inventors have now determined that if the functionality of the small spacer molecule is covalently incorporated into the reservoir molecules described previously, such that a single molecule is formed, then improvements in stability and reproducibility of the membrane formation, as well as improved ionophore conduction can be achieved. Additionally the manufacture of the membrane is simplified as fewer components are required.
Accordingly, in a first aspect, the present invention consists in a linker lipid for use in attaching a membrane including a plurality of ionophores to an electrode and providing a space between the membrane and the electrode in which the membrane is either in part or totally made up of the linker lipid, the linker lipid comprising within the same molecule a hydrophobic region capable of spanning the membrane, an attachment group used to attach the molecule to an electrode surface, a hydrophilic region intermediate said hydrophobic region and the attachment group, and a polar head group region attached to the hydrophobic region at a site remote from the hydrophilic region wherein said attachment group has a cross sectional area that is at least two times the cross sectional area of the hydrophilic region.
It is preferred that the head group, hydrophobic region, and hydrophilic region are as described previously in WO 92/17788 and WO 94/07593. The linker lipid in this case may be wholly synthetic or derived from naturally occurring membrane spanning lipids or archaebacterial lipids.
The hydrophilic region of the linker lipid is preferably a long chain hydrophilic compound. The hydrophilic region of the linker lipid may be composed of oligo/poly ethers, oligo/poly peptides, oligo/poly amides, oligo/poly amines, oligo/poly esters, oligo/poly saccharides, polyols, multiple charged groups (positive and/or negative), electroactive species or combinations thereof. The main requirement of the hydrophilic region of the linker lipid is that it allows the diffusion of ions through the ionophores provided in the membrane. This is achieved by the placement of suitable ion and/or water binding sites along or within the length of the long chain that makes up the reservoir region.
In a preferred embodiment of the invention the hydrophilic region consists of an oligoethylene oxide group. The oligoethylene oxide group may consist of four to twenty ethylene oxide units.
In a further preferred embodiment the hydrophilic region consists of a subunit of tetraethylene glycol attached to succinic acid. This tetraethylene glycol/succinic acid subunit may be repeated 1-4 times.
In a further preferred embodiment the hydrophilic region is formed by group transfer or anionic polymerisation of suitable monomers.
In a further preferred embodiment the hydrophilic region consists of mercaptoethanol, succinic acid, 1,4-diesterified 1,2,3,4-butanetetraol and succinic acid subunits. The succinic acid/1,4-diesterified 1,2,3,4-butanetetraol may be repeated 1-4 times.
In yet another embodiment the hydrophilic region may consist of an oligopropylene glycol of between 1 to 20 propylene glycol units in length. It is further preferred that the hydrophilic region consists oligopropylene glycols of between 2 and 8 propylene glycol units that are functionalised at each end with an N-alkyl amine functionality and that may be joined together via acid units forming tertiary amides.
It is further preferred that the hydrophilic region consists of oligoethylene glycols of between 2 and 10 ethylene glycol units that are functionalised at each end with an N-alkyl amine functionality and that may be joined together via acid units forming tertiary amides.
In a preferred embodiment of the present invention the head group of the linker lipid comprises a receptor reactive with an analyte or a group capable of attaching to a protein receptor.
In a preferred embodiment, the head group comprises a biotin or biotin derivative capable of complexing streptavidin, avidin or one of the common biotin binding proteins.
In a further preferred embodiment the biotin group is linked to the linker lipid via 1 to 8 aminocaproyl groups.
In a further preferred embodiment two biotin groups are attached to the linker lipid such that both biotin groups are capable of complexing a single avidin or streptavidin molecule so as to increase the overall complexing ability and strength of the linker lipid to the avidin or streptavidin.
In a further preferred embodiment of the present invention the hydrophobic region of the membrane spanning lipid comprises a hydrocarbon backbone of between 20-60 angstroms in length with sites of attachment at either end of the hydrocarbon backbone to which are attached at least two hydrocarbon side chains such as phytanyl chains.
In a further preferred embodiment of the present invention the hydrophobic region of the membrane spanning lipid comprises a hydrocarbon backbone of between 20-60 angstroms in length with sites of attachment at either end of the hydrocarbon backbone to which are attached at one end zero or one hydrocarbon sidechain and at least two to four hydrocarbon sidechains at the other end.
The hydrocarbon backbone may comprise a straight methylene chain hydrocarbon, or a hydrocarbon chain optionally substituted with additional groups selected from alkyl, aryl, ether and amine groups, or may comprise two shorter hydrocarbon chains that have been joined via ether, amine, or biphenyl ether groups. Those skilled in the art will appreciate that other functionalities that can link two hydrocarbon chains may also be employed.
It is preferred that the means by which the hydrocarbon chains are attached to the hydrocarbon backbone is via a polyhydroxylated hydrocarbon containing from 3 to 20 hydroxyl groups.
It is further preferred that the means by which the hydrocarbon sidechains are attached to the hydrocarbon backbone is via glycerol, glutamic acid, erythritol, threitol or pentaerythritol groups.
It is preferred that the length of the hydrocarbon sidechains are approximately half the total length of the hydrocarbon backbone.
It is further preferred that the hydrocarbon sidechains are phytanyl chains.
It is further preferred that the hydrocarbon sidechains are mono- or per-methylated hydrocarbon chains or a hydrocarbon chain optionally substituted with additional groups selected from alkyl, aryl, ether and amine groups.
It is preferred that for the case of the electrode material being a gold, platinum, palladium, silver or other coinage metal substrate or combination thereof, the attachment region includes sulfur containing groups such as thiols, disulfides, sulfides thiocyanates. However as previously described, other groups such as organosilanes that form strong attachment to a variety of conductive substrates may also be used.
In the case where the hydrophilic region of the linker lipid is a single chain it is preferred that the attachment region of the molecule is an array containing two to twenty sulfur atoms.
It is further preferred that the attachment region includes between one to three disulfide groups.
Further preferred that the attachment region includes up to 6 thiol groups.
It is further preferred that the attachment group has the following structure:
Xxe2x80x94Y[(CH2)nSR]m
where X is either a carbon, nitrogen or oxygen to which the hydrophilic region is attached, Y is a carbon or if X is a carbon Y may be a nitrogen, n is between 1 to 6, m is between 1 to 3 if Y is a carbon and between 1 to 2 if Y is a nitrogen, and R is a small group such as any of the following xe2x80x94SH, xe2x80x94SCH2Ph, xe2x80x94SCH2CO2H, xe2x80x94SCH2CH2CO2H, xe2x80x94SCH2CH2OH, xe2x80x94SCH2CH2CH2OH, xe2x80x94SCH3, xe2x80x94SCH2CH3, xe2x80x94SCH2CH2CH3, xe2x80x94SCH2CO2CH3, xe2x80x94SCH2CO2CH2CH3, an a containing between 1 and 4 carbon atoms, or an aryl group.
In a further preferred embodiment of the present invention the attachment group has the following structure:
Xxe2x80x94Y[(CH2)nZ(CH2)pSR]m
where X is either a carbon, nitrogen or oxygen to which the hydrophilic region is attached, Y is a carbon or if X is a carbon Y may be a nitrogen, m is between 1 to 3 if Y is a carbon and between 1 to 2 if Y is a nitrogen, where Z is O, NH, NR1, an amide or ketone, and where n is between 1 and 5 and p is between 2 and 5, unless Y is N, in which case n is between 2 and 5, and where R1 is an alkyl chain containing between 1 and 4 carbon atoms, and R is a small group such as any of the following xe2x80x94SH, xe2x80x94SCH2Ph, xe2x80x94SCH2CO2H, xe2x80x94SCH2CH2CO2H, xe2x80x94SCH2CH2OH, xe2x80x94SCH2CH2CH2OH, xe2x80x94SCH3, xe2x80x94SCH2CH3, xe2x80x94SCH2CH2CH3, xe2x80x94SCH2CO2CH3, xe2x80x94SCH2CO2CH2CH3, an alkyl chain containing between 1 and 4 carbon atoms, or an aryl group.
It is further prefered that the attachment group is thiooctic acid or bis-thiooctic acid derivative.
It is further preferred that the attachment group is the cyclic oxidised form of dithiothreitol.
It is further preferred that the attachment group contains one to three bis(4-hydroxymethyl)-1,2-dithiacyclopentane groups.
It is further preferred that the attachment group contains a bis(4-hydroxymethyl)-1,2-dithiacyclopentane groups where the hydrophilic reservoir is attached via one of the 4-hydroxymethyl moieties of the bis(4-hydroxymethyl)-1,2-dithiacyclopentane and where the other 4-hydroxymethyl moiety may be the hydroxy functionality or may have been further functionalised to a methyl ether, ethyl ether, propyl ether, acetate, or succinate, or a group of the formula (CH2)nCOZ where n is 0 to 4, and Z is OR, or NR1R2, where R, R1 and R2 are independently hydrogen or alkyl chains containing between 1 and 4 carbon atoms.
It is further preferred that the attachment group contains a bis(4-hydroxymethyl)-1,2-dithiacyclopentane groups where the hydrophilic reservoir is attached via one of the 4-hydroxymethyl moieties of the bis(4-hydroxymethyl)-1,2-dithiacyclopentane and where the other 4-hydroxymethyl moiety may be linked to between one and three other bis(4-hydroxymethyl)-1,2-dithiacyclopentane groups.
It is further preferred that the linking group be ethyleneoxy or diethyleneoxy.
It is further preferred that the attachment group contains one to three dithiothreitol groups.
It is further preferred that the attachment group contains a trans-4,5-dihydroxy-1,2-dithiacyclohexane groups where the hydrophilic reservoir is attached via one of the 4,5-hydroxy moieties of the trans-4,5-dihydroxy-1,2-dithiacyclohexane and where the other 4,5-hydroxy moiety may be the hydroxy functionality or may have been further functionalised to a methyl ether, ethyl ether, propyl ether, acetate, or succinate, or a group of the formula (CH2)nCOZ where n is 0 to 4, and Z is OR, or NR1R2, where R, R1 and R2 are independently hydrogen or alkyl chains containing between 1 and 4 carbon atoms.
It is further preferred that the attachment group contains a trans-4,5-dihydroxy-1,2-dithiacyclohexane groups where the hydrophilic reservoir is attached via one of the 4,5-hydroxy moieties of the trans-4,5-dihydroxy-1,2-dithiacyclohexane and where the other 4,5-hydroxy moiety may be linked to between one and three other trans-4,5-dihydroxy-1,2-dithiacyclohexane groups.
In a further preferred embodiment the cross sectional area of the hydrophobic region is similar to the cross sectional area of the attachment group as shown schematically in FIG. 1.
In a second aspect, the present invention consists in a linker lipid for use in attaching a bilayer membrane including a plurality of ionophores to an electrode and providing a space between the membrane and the electrode in which the membrane layer proximate the electrode is either in part or totally made up of the linker lipid, the linker lipid comprising within the same molecule a hydrophobic region which spans half the membrane, an attachment group used to attach the molecule to an electrode surface, and a hydrophilic region intermediate said hydrophobic region and the attachment group, wherein said attachment group has a cross sectional area that is at least two times the cross sectional area of the hydrophilic region.
It is preferred that the hydrophobic region is a phytanyl chain.
It is further preferred that the hydrophobic region is a mono- or per-methylated hydrocarbon chain or a hydrocarbon chain optionally substituted with additional groups selected from alkyl, aryl, ether and amine groups.
It is preferred that the hydrophobic region is comprised of a polyether containing hydrocarbon chains, such as phytanyl, attached to polyol.
It is further preferred that the hydrophobic region comprises 2 to 4 hydrocarbon chains such as phytanyl chains.
It is further preferred that the hydrophobic region comprise a diphytanyl glyceryl ether.
It is further preferred that the hydrophobic region comprise a triphytanyl pentaerythrityl ether.
It is further preferred that the hydrophobic region comprise a triphytanyl threityl ether.
It is further preferred that the hydrophobic region comprise a triphytanyl erythritol ether.
It is preferred that for the case of the electrode material being a gold, platinum, palladium, silver or other coinage metal substrate or combination thereof, the attachment region includes sulfur containing groups such as thiols, disulfides, sulfides thiocyanates. However as previously described, other groups such as organosilanes that form strong attachment to a variety of conductive substrates may also be used.
In the case where the hydrophilic region of the linker lipid is a single chain it is preferred that the attachment region of the molecule is an array containing two to twenty sulfur atoms.
It is further preferred that the attachment region includes between one to three disulfide groups.
Further preferred that the attachment region includes up to 6 thiol groups.
It is further preferred that the attachment group has the following structure:
Xxe2x80x94Y[(CH2)nSR]m
where X is either a carbon, nitrogen or oxygen to which the hydrophilic region is attached, Y is a carbon or if X is a carbon Y may be a nitrogen, n is between 1 to 6, m is between 1 to 3 if Y is a carbon and between 1 to 2 if Y is a nitrogen, and R is a small group such as any of the following xe2x80x94SH, xe2x80x94SCH2Ph, xe2x80x94SCH2CO2H, xe2x80x94SCH2CH2CO2H, xe2x80x94SCH2CH2OH, xe2x80x94SCH2CH2CH2OH, xe2x80x94SCH3, xe2x80x94SCH2CH3, xe2x80x94SCH2CH2CH3, xe2x80x94SCH2CO2CH3, xe2x80x94SCH2CO2CH2CH3, an alkyl chain containing between 1 and 4 carbon atoms, or an aryl group.
In a further preferred embodiment of the present invention the attachment group has the following structure:
Xxe2x80x94Y[(CH2)nZ(CH2)pSR]m
where X is either a carbon, nitrogen or oxygen to which the hydrophilic region is attached, Y is a carbon or if X is a carbon Y may be a nitrogen, m is between 1 to 3 if Y is a carbon and between 1 to 2 if Y is a nitrogen, where Z is O, NH, NR1, an amide or ketone, and where n is between 1 and 5 and p is between 2 and 5, unless Y is N, in which case n is between 2 and 5, and where R1 is an alkyl chain containing between 1 and 4 carbon atoms, and R is a small group such as any of the following xe2x80x94SH, xe2x80x94SCH2Ph, xe2x80x94SCH2CO2H, xe2x80x94SCH2CH2CO2H, xe2x80x94SCH2CH2OH, xe2x80x94SCH2CH2CH2OH, xe2x80x94SCH3, xe2x80x94SCH2CH3, xe2x80x94SCH2CH2CH3, xe2x80x94SCH2CO2CH3, xe2x80x94SCH2CO2CH2CH3, an alkyl chain containing between 1 and 4 carbon atoms, or an aryl group.
It is further prefered that the attachment group is thiooctic acid or bis-thiooctic acid derivative.
It is further preferred that the attachment group is the cyclic oxidised form of dithiothreitol.
It is further preferred that the attachment group contains one to three bis(4-hydroxymethyl)-1,2-dithiacyclopentane groups.
It is further preferred that the attachment group contains a bis(4-hydroxymethyl)-1,2-dithiacyclopentane groups where the hydrophilic region is attached via one of the 4-hydroxymethyl moieties of the bis(4-hydroxymethyl)-1,2-dithiacyclopentane and where the other 4-hydroxymethyl moiety may be the hydroxy functionality or may have been further functionalised to a methyl ether, ethyl ether, propyl ether, acetate, or succinate. or a group of the formula (CH2)nCOZ where n is 0 to 4, and Z is OR, or NR1R2, where R, R1 and R2 are independently hydrogen or alkyl chains containing between 1 and 4 carbon atoms.
It is further preferred that the attachment group contains a bis(4-hydroxymethyl)-1,2-dithiacyclopentane groups where the hydrophilic region is attached via one of the 4-hydroxymethyl moieties of the bis(4-hydroxymethyl)-1,2-dithiacyclopentane and where the other 4-hydroxymethyl moiety may be linked to between one and three other bis(4-hydroxymethyl)-1,2-dithiacyclopentane groups.
It is further preferred that the linking group be ethyleneoxy or diethyleneoxy.
It is further preferred that the attachment group contains one to three dithiothreitol groups.
It is further preferred that the attachment group contains a trans-4,5-dihydroxy-1,2-dithiacyclohexane groups where the hydrophilic region is attached via one of the 4,5-hydroxy moieties of the trans-4,5-dihydroxy-1,2-dithiacyclohexane and where the other 4,5-hydroxy moiety may be the hydroxy functionality or may have been further functionalised to a methyl ether, ethyl ether, propyl ether, acetate, or succinate, or a group of the formula (CH2)nCOZ where n is 0 to 4, and Z is OR, or NR1R2, where R, R1and R2 are independently hydrogen or alkyl chains containing between 1 and 4 carbon atoms.
It is further preferred that the attachment group contains a trans-4,5-dihydroxy-1,2-dithiacyclohexane groups where the hydrophilic region is attached via one of the 4,5-hydroxy moieties of the trans-4,5-dihydroxy-1,2-dithiacyclohexane and where the other 4,5-hydroxy moiety may be linked to between one and three other trans-4,5-dihydroxy-1,2-dithiacyclohexane groups.
Ionophore ion channels such as gramicidin generally need to assume a particular conformation in order to form conducting channels. Gramicidin A for instance is thought to assume a beta-helical structure in its conducting form. It is thought that if there is sufficient crowding of the linker gramicidin by linker lipid molecules during the deposition of the inner sulfur/gold lipid layer, then this crowding may adversely affect the ability of the ion channel to assume its proper conformation and hence reduce its capability of forming conducting channels. Previously this crowding was thought to be minimised by the use of small sulfur containing spacer molecules. A more controllable method is to increase the cross sectional area of the attachment group such that its cross sectional area is comparable to the cross sectional area of the ion channel.
Hence, in a third aspect, the present invention consists in a linker ion channel for use in a bilayer or monolayer membrane based biosensor including an electrode, said linker ion channel comprising within the same molecule a hydrophobic ion channel which spans at least half the membrane, an attachment group to attach the linker ion channel to the electrode surface, a hydrophilic region intermediate said hydrophobic ion channel and the attachment group, wherein said attachment group has a cross sectional area that is at least the cross sectional area of the hydrophobic ion channel.
It is preferred that the ion channel is gramicidin or one of its derivatives.
It is further preferred that the ion channel is a synthetic ion channel.
It is preferred that for the case of the electrode material being a gold, platinum, palladium, silver or other coinage metal substrate or combination thereof, the attachment region includes sulfur containing groups such as thiols, disulfides, sulfides thiocyanates. However as previously described, other groups such as organosilanes that form strong attachment to a variety of conductive substrates may also be used.
In the case where the hydrophilic region of the linker lipid is a single chain it is preferred that the attachment region of the molecule is an array containing two to twenty sulfur atoms.
It is further preferred that the attachment region includes between one to three disulfide groups.
Further preferred that the attachment region includes up to 6 thiol groups.
It is further preferred that the attachment group has the following structure:
Xxe2x80x94Y[(CH2)nSR]m
where X is either a carbon, nitrogen or oxygen to which the hydrophilic region is attached, Y is a carbon or if X is a carbon Y may be a nitrogen, n is between 1 to 6, m is between 1 to 3 if Y is a carbon and between 1 to 2 if Y is a nitrogen, and R is a small group such as any of the following xe2x80x94SH, xe2x80x94SCH2Ph, xe2x80x94SCH2CO2H, xe2x80x94SCH2CH2CO2H, xe2x80x94SCH2CH2OH, xe2x80x94SCH2CH2CH2OH, xe2x80x94SCH3, xe2x80x94SCH2CH3, xe2x80x94SCH2CH2CH3, xe2x80x94SCH2CO2CH3, xe2x80x94SCH2CO2CH2CH3, an alkyl chain containing between 1 and 4 carbon atoms, or an aryl group.
In a further preferred embodiment of the present invention the attachment group has the following structure:
Xxe2x80x94Y[(CH2)nZ(CH2)pSR]m
where X is either a carbon, nitrogen or oxygen to which the hydrophilic region is attached, Y is a carbon or if X is a carbon Y may be a nitrogen, m is between 1 to 3 if Y is a carbon and between 1 to 2 if Y is a nitrogen, where Z is O, NH, NR1, an amide or ketone, and where n is between 1 and 5 and p is between 2 and 5, unless Y is N, in which case n is between 2 and 5, and where R1is. an alkyl chain containing between 1 and 4 carbon atoms, and R is a small group such as any of the following xe2x80x94SH, xe2x80x94SCH2Ph, xe2x80x94SCH2CO2H, xe2x80x94SCH2CH2CO2H, xe2x80x94SCH2CH2OH, xe2x80x94SCH2CH2CH2OH, xe2x80x94SCH3, xe2x80x94SCH2CH3, xe2x80x94SCH2CH2CH3, xe2x80x94SCH2CO2CH3, xe2x80x94SCH2CO2CH2CH3, an alkyl chain containing between 1 and 4 carbon atoms, or an aryl group.
In a further preferred embodiment of the present invention the attachment group has the following structure:
Pxe2x80x94Q[(CH2)nT(CH2)pW]m
where P either a carbon, nitrogen or oxygen to which the hydrophilic reservoir region is attached, Q is a carbon or if P is a carbon Q may be a nitrogen, n is between 1 to 6, m is between 1 to 3 if Q is a carbon and between 1 to 2 if Q is a nitrogen, T is O, NH, NR1, an amide or ketone, and where n is between 1 and 5 and p is between 2 and 5, unless Q is N, in which case n is between 2 and 5, and where R1 is an alkyl chain containing between 1 and 4 carbon atoms, and W is a group of the formula:
Xxe2x80x94Y[(CH2)nZ(CH2)pSR]m
where X is either a carbon, nitrogen or oxygen, Y is a carbon or an alkyl chain of 1-4 carbons or if X is a carbon Y may be a nitrogen, m is between 1 to 3 if Y is a carbon and between 1 to 2 if Y is a nitrogen, where Z is a bond, O, NH, NR1, an amide or ketone, and where n is between 1 and 5 and p is between 2 and 5, unless Y is N, in which case n is between 2 and 5, and where R1is an alkyl chain containing between 1 and 4 carbon atoms, and R is a small group such as any of the following xe2x80x94SH, xe2x80x94SCH2Ph, xe2x80x94SCH2CO2H, xe2x80x94SCH2CH2CO2H, xe2x80x94SCH2CH2OH, xe2x80x94SCH2CH2CH2OH, xe2x80x94SCH3, xe2x80x94SCH2CH3, xe2x80x94SCH2CH2CH3, xe2x80x94SCH2CO2CH3, xe2x80x94SCH2CO2CH2CH3, an alkyl chain containing between 1 and 4 carbon atoms, or an aryl group.
It is further prefered that the attachment group is thiooctic acid or bis-thiooctic acid derivative.
It is further preferred that the attachment group is the cyclic oxidised form of dithiothreitol.
It is further preferred that the attachment group contains one to three bis(4-hydroxymethyl)-1,2-dithiacyclopentane groups.
It is further preferred that the attachment group contains a bis(4-hydroxymethyl)-1,2-dithiacyclopentane groups where the hydrophilic region is attached via one of the 4-hydroxymethyl moieties of the bis(4-hydroxymethyl)-1,2-dithiacyclopentane and where the other 4-hydroxymethyl moiety may be the hydroxy functionality or may have been further functionalised to a methyl ether, ethyl ether, propyl ether, acetate, or succinate, or a group of the formula (CH2)nCOZ where n is 0 to 4, and Z is OR, or NR1R2, where R, R1and R2 are independently hydrogen or alkyl chains containing between 1 and 4 carbon atoms.
It is further preferred that the attachment group contains a bis(4-hydroxymethyl)-1,2-dithiacyclopentane groups where the hydrophilic region is attached via one of the 4-hydroxymethyl moieties of the bis(4-hydroxymethyl)-1,2-dithiacyclopentane and where the other 4-hydroxymethyl moiety may be linked to between one and three other bis(4-hydroxymethyl)-1,2-dithiacyclopentane groups.
It is further preferred that the linking group be ethyleneoxy or diethyleneoxy.
It is further preferred that the attachment group contains one to three dithiothreitol groups.
It is further preferred that the attachment group contains a trans-4,5-dihydroxy-1,2-dithiacyclohexane groups where the hydrophilic region is attached via one of the 4,5-hydroxy moieties of the trans-4,5-dihydroxy-1,2-dithiacyclohexane and where the other 4,5-hydroxy moiety may be the hydroxy functionality or may have been further functionalised to a methyl ether, ethyl ether, propyl ether, acetate, or succinate, or a group of the formula (CH2)nCOZ where n is 0 to 4, and Z is OR, or NR1R2, where R, R1 and R2 are independently hydrogen or alkyl chains containing between 1 and 4 carbon atoms.
It is further preferred that the attachment group contains a trans-4,5-dihydroxy-1,2-dithiacyclohexane groups where the hydrophilic region is attached via one of the 4,5-hydroxy moieties of the trans-4,5-dihydroxy-1,2-dithiacyclohexane and where the other 4,5-hydroxy moiety may be linked to between one and three other trans-4,5-dihydroxy-1,2-dithiacyclohexane groups.
Sulfur containing compounds can be prepared by conventional literature procedures. Cyclic disulfides can also be prepared by conventional literature procedures, however, it is presently preferred that cyclic disulfides are prepared by the cyclisation of a xcex1,xcfx89-disubstituted thiocyanates by treatment with a source of fluoride ion. It is further preferred that the fluoride ion source is tetrabutylammonium fluoride. The reaction is conducted in an organic solvent or mixture of solvents, at a temperature between xe2x88x9270xc2x0 and 100xc2x0 C. It is further preferred that the reaction is conducted in aqueous tetrahydrofuran between 0xc2x0 and 50xc2x0 C.
Accordingly in a fourth aspect the present invention consists in a method of producing cyclic disulfides, the method comprising reacting an xcex1,xcfx89-disubstituted thiocyanate with a source of fluoride ion.
In a fifth aspect the present invention consists in linker lipids described in the first aspect of the invention which in addition have an ionophore covalently attached to the hydrophobic region of the linker lipid via at least one tethering chain which is long enough such that the attached ionophore may traverse the membrane in such a way that it is still able to transport ions across the membrane.
Typical ionophores may be natural, semi-synthetic or wholly synthetic ionophores such as valinomycin, nonactin, crown ether derivatives, podands, coronands, cryptands, gramicidin.
In a sixth aspect the present invention consists in a membrane formed exclusively from linker lipids of the first aspect of the invention and fourth aspect of the present invention to which are tethered ionophores.
In a seventh aspect the present invention consists in a membrane formed from a plurality of linker lipids according to the first aspect and a plurality of linker lipids according to the second aspect of the invention and additional lipids and ion channels so as to form a membrane that has the similar thickness to a normal bilayer membrane structure.
It is further preferred that the ion channel added to the membrane is a gramicidin derivative that is capable of being linked to a protein such as streptavidin or an antibody or antibody fragment or other receptor molecule.
In a preferred embodiment of this aspect of the present invention the membrane also includes a plurality of linker ion channels according to the third aspect of the invention.
As will be appreciated by those skilled in this field that the membranes of the present invention may include additional lipid. In these instances it is preferred that the additional lipid is a mixture of diphytanyl ether phosphatidyl choline and glycerol diphytanyl ether in a ratio of between 9:1 to 6:4.
Those skilled in the art will also appreciate that where molecules can exist as stereoisomers, that any of the individual stereoisomers, or mixtures thereof, may be employed. In addition, where amines are employed it will be appreciated that common salts of the amines could also be employed.